B Material and Methods
6. Cell culture
6.1 Cell culture media Growth Medium:
Modified eagle medium (MEM, PAA) or Dulbecco’s modified eagle medium (DMEM, PAA), supplemented with 10% (v/v) fetal bovine serum (FBS, PAA). For liver cell lines, non-essential amino acids (PAA) were added.
Freezing Medium:
DMEM or MEM supplemented with 10% (v/v) FBS and filter sterilised dimethylsulfoxide (DMSO).
Phosphate Buffered Saline (PBS) (PAA):
137 mM NaCl 2.6 mM KCl 10 mM Na2HPO4
1.8 mM KH2PO4
Trypsin-EDTA Solution (PAA)
0.53 mM tetra-sodium ethylene diamine tetra-acetic acid (EDTA) 0.05% trypsin
6.1.1 Cultivation of eukaryotic cell lines
All cells were grown in a humidified incubator at 37°C and 5% CO2.
Human embryonic kidney 293 cells (ATCC CRL-1573) and HeLa cells (ATCC CCL-2) were grown in MEM supplemented with 10% FBS.
Huh7, Sk-Hep-1 (ATCC HTB 52) and murine Hepa 1A cells were kept in DMEM containing 10% FBS and 0.1 mM non-essential amino acids.
116 cells (Palmer and Ng 2003) were used for production of GD vectors. This cell line carries a hygromycin resistance gene, expresses Cre recombinase, and was grown in MEM supplemented with 10% FBS and 100 µg/ml hygromycin B.
W162 cells (ATCC CRL-2783), a Vero cell derivative stably expressing the E4 region of Ad5, supports the growth of adenoviral E4 deletion mutants. This cell line was maintained in MEM with 2 mM L-glutamine, adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate and 10% FBS.
293M cells were grown in MEM supplemented with 10% FBS and 100 µg/ml hygromycin for selection of PaeR7 methylase expressing cells. 293M cells were generated in the present study.
116M cells were routinely grown with DMEM supplemented with 10% FBS, 100 µg/ml hygromycin (PAA) for selection of Cre expression and 575 ng/ml puromycin (PAA) for selection of PaeR7 methylase expression. 116M cells were generated in the present study.
6.1.2 Establishment of cell cultures from cryostocks
The thawed cell suspension was transferred to a tube containing growth medium. Cells were collected by centrifugation at 200 g for 3 min at room temperature. The growth medium was removed, the cell pellet was re-suspended with 1 ml of fresh growth medium and transferred to cell culture dishes containing the appropriate growth medium.
6.1.3 Preparation of permanent cell culture stocks in liquid nitrogen
Cells were grown to at least 50% confluency. Cells were trypsinised and collected by centrifugation at 200 g for 3 min at room temperature. The supernatant was removed and the cell pellet was re-suspended in 3 ml of freezing medium. 500 µl of the cell suspension were aliquoted into cryovials and kept in liquid nitrogen for long-term storage.
6.1.4 Passaging of eukaryotic cell lines
At a concluence of more than 50%, cells were split at a 1:10 ratio. Cells were removed from the dish with trypsin-EDTA solution (PAA). Trypsinised cells were immediately transferred to new cell culture plates containing the appropriate growth medium.
6.1.5 Establishment of stable eukaryotic cell lines
Plasmids were transfected into 116 and 293 cells using Superfect transfection reagent according to the manufacturer’s instructions. Transfected cells were incubated with the respective antibiotics. After 20 days of incubation under selection pressure, single cell colonies were isolated. Cells were further expanded in the respective growth medium.
6.2 Gene-deleted adenoviral vector hFIX (GD AdV)
GD AdV hFIX was mainly used for the present study. GD AdV AdFTC-hFIX-lucRNAi was based on the parental plasmid pAdFTC (Ehrhardt and Kay 2002) and contains Ad5 5’- and 3’- inverted terminal repeats (ITR) and a packaging signal (Ψ). The stuffer DNA of this vector consists of the matrix attachment region (MAR) of the murine immunoglobulin κ locus and alphoid repeat DNA sequences from human chromosome 17. The alphoid repeat DNA is equivalent with a centromeric region and contains potential mammalian origins of replication.
The human blood coagulation factor IX (hFIX) expression cassette was described previously (Miao et al. 2000) and contained two liver specific enhancers - apolipoprotein E (ApoE) enhancer and the hepatic control region (HCR) - and a human alpha-1-antitrypsin promoter (hAAT-p) controlling expression of the hFIX minigene. Additionally, this GD AdV contained an expression cassette encoding small hairpin RNA (shRNA) against firefly luciferase driven by human U6 snRNA promoter (hU6-p) [figure 4]. The capsid of this vector is identical to that of Ad5 wt.
5´-ITR Ψ IgκMAR alphoid repeat DNA ApoE/HCR hAAT-p hFIX minigene ITR -3´
GD AdV hFIX
5´-ITR Ψ IgκMAR alphoid repeat DNA ApoE/HCR hAAT-p hFIX minigene ITR -3´
GD AdV hFIX
Figure 4: Schematic representation of the genome of gene-deleted adenoviral vector hFIX (GD AdV hFIX). For description of the single elements see text.
6.3 Large-scale production and purification of viral vectors and viruses 6.3.1 Pre-amplification steps
The linearised GD AdV DNA construct was transfected into the producer cell line 116 according to the manufacturer’s instructions (Superfect, Qiagen). Sixteen to 18 h post transfection, co-infect with helper virus at an MOI of 5. Cells and supernatant (= passage P0) were harvested 48 h post-infection and frozen at −80°C. Now, 3 serial passaging steps (P1 - P3) on 6 cm, 10 cm and 15 cm dishes are required for pre-amplification of GD AdV. Usually, one third of the freezed-thawed lysate of the previous passage is applied to infect the next plate. Co-infection with helper virus was always at an MOI of 5. Lysate was generally harvested when complete cytopathic effect was observed.
6.3.2 Large-scale production of GD AdV
Large-scale GD AdV production was performed as described earlier (Parks et al. 1996;
Palmer and Ng 2003; Jager et al. 2008 submitted; figure 5). In brief, 10 confluent 15 cm dishes of 116 cells were transferred into 1 litre of MEM (Gibco) supplemented with 10% FBS (Sigma) and 100 µg/ml hygromycin B (Invitrogen). For production of methylated GD AdV, 116M cells growing with 575 ng/ml puromycin were used. Over the next days, the same growth medium with all necessary supplementals was added to a final volume of 3 litres (500 ml on days 2 and 3, 1000 ml on day 4). On day five, 3 litres of 116 cells at 3 to 4x105 cells/ml were harvested by centrifugation (8 min at 1500 rpm in a clinical centrifuge), resuspended in 120 ml volume medium and co-infected with 100% of the crude lysate from the 15 cm dish of serial passaging step 3 and AdNG163R-2 helper virus (Palmer and Ng 2003) at an MOI of 1 PFU/cell. In an alternative simplified protocol, purified GD AdV instead of crude lysate was used as inoculum at an MOI of 100 for co-infection of 3 litres of 116 cells.Virus adsorption was performed at 37°C on a magnetic stir plate for 2 h, after which medium (MEM supplemented with 5% FBS, Sigma) was added to a final volume of 2 litres. Co-infected cells were harvested 48 h later for lysis via centrifugation for 10 min at 1500 rpm in a clinical
centrifuge and were re-suspended in a total volume of 30 ml PBS. Subsequently, GD AdV virions were purified.
ITR ITR
5´- -3´
∆E3 loxP loxPΨ
early and late genes
ITR
5´-Ψ
ITR -3´
„stuffer“ DNA Transgene
∆E1
ITR ITR
5´- -3´
∆E3 loxP loxPΨ
early and late genes
ITR
5´-Ψ
ITR -3´
„stuffer“ DNA Transgene
∆E1
Figure 5: Large-scale production of GD AdV. The helper virus provides all necessary genes for GD AdV production in trans. Helper virus genomes are not efficiently packaged due to excision of their packaging signal Ψ in Cre-expressing 116 cells. This method results in high titres of GD AdV. In the upper part of the figure, all relevant characteristics of the helper virus and the GD AdV are indicated.
6.3.3 Large-scale production of Ad5 wt, ∆E4 mutants and 1st generation vector
Ad5 ∆E4 mutants (H5dl1004 lacking nucleotides 2845 to 981 of E4) were amplified in W162 trans-complementing cells and Ad5 wild type was amplified in adherent 293 or 293M cells. Amplification of the E1 and E3 deleted recombinant 1st generation adenovirus (∆E1/E3 AdV) was performed in 293 cells. All of these viruses/vectors were produced on 15-30 confluent 15 cm cell culture plates. Viral particles were purified using CsCl density centrifugation (see also 6.3.5).
6.3.4 Purification of adenoviruses and adenoviral vectors
Cells and supernatant of infected dishes were collected in conic 500 ml bottles. Cells containing the virus were spun down for 8 min at 200 x g. Supernatant was removed and the cell pellet was resuspended in 14-28 ml DPBS. This suspension can be stored at -80°C. To
GD AdV packaged Helper virus not packaged
Helper virus AdNG163R-2 GD AdV
Cre-loxP recombination 116 Cre-expressing cells
release the virus from the cells, the suspension was four times freezed in liquid nitrogen and thawed in a 37°C waterbath. The lysate was spun down for 8 min at 200 x g and the supernatant containing the virus was collected.
For purification of adenovirus preparations, the suspension obtained above was loaded onto one step and one continuous gradient. The step gradient was established by pipetting CsCl solutions with different densities in the following order:
0.5 ml 1.5 g/cm3 CsCl 3 ml 1.35 g/cm3 CsCl 3.5 ml 1.25 g/cm3 CsCl 5 ml virus/vector
This step gradient was spun for 1.5 h at 35000 rpm (226000 x g)/ 12°C in an ultracentrifuge.
The lower bluish band containing the virus was collected and mixed with 1.35 g/cm3 CsCl (final volume 12 ml). This continuous gradient was spun for at least 20 h at 35000 rpm (226000 x g)/ 12°C. The virus band was collected in an as little volume as possible. The purified virus was dialysed over night at 4°C in 2 l dialysis solution (10 mM Tris (pH 7.5, 10% Glycerol, 1 mM MgCl2) at very low stir speed and aliquotted.
6.4 Adenovirus DNA analysis
Fifty µl CsCl-purified virus were incubated in 150 µl lysis buffer (10 mM Tris, 10 mM EDTA, 0,5% SDS) supplemented with 10 µl proteinase K (Qiagen, 600 mAU/ml = 20 mg/ml) for 2 h at 55°C. Subsequently, samples were purified with phenol:chloroform:isoamylalcohol.
Samples were centrifuged 2 min at full speed in a bench top centrifuge and upper phase was transferred to a new tube. DNA was precipitated with ice cold 100% ethanol for 20 min at -80°C and centrifugation at full speed and 4°C for 10 min. Precipitated DNA was resuspended in 50 µl water. For DNA analysis, 15 µl DNA were digested with restriction endonuclease for 1.5 h.
6.5 Titering of adenoviral vector preparations 6.5.1 Determination of the physical titre
Viral DNA was released from virions obtained from CsCl gradients in TE buffer (10 mM Tris, 10 mM EDTA) with 0.1% SDS. The A260 was measured to determine the viral titre.
By the well-characterized absorption of pure adenovi rus at 260 nm (Maizel et al. 1968), an OD260 of 0.01 corresponds to a viral particle concentration of 1.1 x 109 VP/ml based on a 36
kb size of the wild type particle genome. Thus, total vi ral particles for GD AdV and other adenovi ruses were calculated as follows: VP/ml x (OD260 x dilution factor x 36 kb)/(size of the vector [kb] x 9.09 x 10–13); where 9.09 x 10–13 is the extinction coefficient for wild-type adenovi rus.
475 µl of measurement buffer (0.1 M TE, 10 mM EDTA, 0.1% SDS) were added to a 25 µl aliquot of the CsCl purified virus. Suspensions were vigorously shaken at 1200 rpm for 15 min in a table top shaker. Subsequently, samples were briefly centrifuged and subjected to OD260nm measurement. Fifty µl of each of 5 aliquots were used for determination.
6.5.2 Determination of the infectious titre by Southern blot
To control that similar amounts of ∆E4 and GD AdV genomes were administered, 293 cells were infected with either virus at OD260 MOIs of 1, 11 and 33. Cells were harvested after 4 h of incubation with trypsin. Thirty µg of total DNA were analysed on a Southern blotted gel. The intensity of the respective band indicates the relative infectious titres. We found that ∆E4 and GD AdV show similar titres at the respective MOI [Appendix figure 3].
6.5.3 Determination of the infectious titre by quantitative real-time PCR
Moreover, the number of total genomes and infectious genomes were determined by quantitative real-time PCR (qPCR) employing the oligonucleotides Rev436 and Ad5ITRfw1-24. For determination of total particles, 10 vp/cell were directly administered to pelleted 293 cells before starting Hirt extraction. Subsequently, total particles were determined analyzing 200 fg of Hirt-extracted DNA in a total volume of 20 µl. Infectious particles were determined analyzing 200 fg of Hirt-extracted DNA of infected (10 vp/cell) 293 cells in a total volume of 20 µl. These infected 293 cells were harvested with trypsin after 4 h of incubation to remove unabsorbed virions. The LightCycler FastStart DNAMasterPlus SYBR Green I (Roche) detected the PCR-products. The programme of the LightCycler (Applied Biosystems) was set as follows: Pre-incubation at 95°C for 10 min, amplification in 45 cycles at 95°C for 10 sec, 55°C for 5 sec, 72°C for 30 sec. All MOI indicated in the present study relate to titre determinations by OD260 measurements [Appendix figure 2].
6.5.4 Determination of the helper virus contamination of GD AdV preparations
The determination of the helper virus conatamination was performed using purified adenoviral DNA. To purify adenoviral DNA from CsCl solutions, three independent adenoviral preparations, each 20 µl, were incubated with 400 µl of proteinase K-SDS solution (proteinase K [0.5 mg/ml], 10 mM Tris-HCl [pH 7.5], 0.5% sodium dodecyl sulfate [SDS], 10 mM EDTA [pH 8]) for 3 h at 37°C. DNA was then precipitated by addition of 1/10 volume of 3 M sodium acetate (pH 5.2) and a 2.5 vol of 95% ethanol, rinsed with 70% ethanol, dried, and resuspended in 25 µl of water. Preparations of GD AdV were evaluated in three different dilutions per assay: 10–3 to 10–5. In a total volume of 20 µl per reaction, 5 µl of the template DNA was added.
Quantitative real-time PCR for helper virus contamination was carried out by the Taqman 7500 Fast Real Time PCR System (Applied Biosystems) amplifying a 235 bp area of the adenoviral late gene 3 (L3). Utilizing the oligonucleotides L3 forward (400 nM) and L3 reverse (400 nM) together with an L3 specific probe (300 nm) (Ella Biotech) the PCR was run with the following programme: AmpErase UNG reaction at 50°C for 2 min, pre-incubation/activation at 95°C for 10 min, amplification and data collection in 40 cycles at 95°C for 15 sec and 60°C for 1 min. Universal Fast PCR Master Mix (Applied Biosystems) was used for Taqman qPCR. Oligonucleotides, probe and protocol parameters were chosen according to Puntel et al. 2006.
The contamination of GD adenoviral vector with helper virus lays at 0.09% compared to the number of total particles [Appendix figure 2]. This amount is well in the range reported by others (Puntel et al. 2007).